Bacteria of the class Mollicutes underwent extreme reduction of genomes and gene expression control systems. Only a few regulators are known to date. In this work, we describe a novel group of transcriptional regulators that are distributed within different Mollicutes and control the expression of restriction-modification systems (RM-systems).Mycoplasma gallisepticum S6. We confirmed our prediction and identified key nucleotides important for C-protein binding. Further we identified novel target promoters of C-protein in M. gallisepticum.We performed cross-species search of putative regulators of RM-systems (C-proteins) and respective binding sites in Mollicutes. We identified a set of novel putative C-protein binding motifs distributed within Mollicutes. We studied the most frequent motif and respective C-protein on the model of M. gallisepticum binds predicted conserved direct repeats of the (GTGTTAN5)2 motif. Apart from its own operon promoter, HsdC can bind to the promoters of the clpB chaperone gene and a tRNA cluster.We found that C-protein of The online version of this article (doi:10.1186/s12866-017-0935-4) contains supplementary material, which is available to authorized users. Mollicutes are wall-less bacteria with a significantly reduced genome. The repertoire of gene expression regulators in Mollicutes is reduced as well. The smallest number of transcription factors is observed within Mycoplasmatales . HoweverMycoplasma gallisepticum as a model organism to study the binding properties of the respective regulator.Restriction-modification systems are widespread in bacteria. They have two enzymatic activities: site-specific DNA methylase and site-specific endonuclease. Generally, RM-systems modify hemimethylated DNA and cleave unmethylated DNA. The majority of RM-systems belong to type I or type II. While all RM-systems consist of restriction (R) and modification (M) subunits, type I systems feature an additional specificity (S) subunit as a separate protein , 3. TypeMycoplasma gallisepticum S6 as a model to study the DNA-binding properties of its C-protein homolog, further termed HsdC (GCW_02350) because in this bacterium it resides within the hsd operon (GCW_02350-GCW_02365). As a result, we identified a novel C-protein binding site.A set of various RM-systems, predominantly of type II, feature specific transcriptional regulators termed controller proteins or C-proteins , 8, whicM. gallisepticum S6 . The amplicon was cloned into the pETm plasmid with C-terminal His-tag as described previously [E. coli BL21-Gold (DE3) cells. Cells were grown overnight, harvested by centrifugation, washed in PBS and lysed with Branson 250 Sonifier (Branson) at 22 kHz for 10 min. The lysate was diluted with sample buffer . The protein was purified on a Tricorn 5/50 column  with Ni Sepharose High Performance  resin using the AKTA FPLC system . After the application of lysate, the column was washed with 25-ml aliquots of sample buffer, then with wash buffer  and finally with elution buffer . After elution, the protein was 60-fold diluted with 20 mM Tris-HCl buffer, pH 7.5 to 20 pmol/μl and directly used for electrophoretic mobility shift assay (EMSA).Cloning and purification procedures were performed as described in . The Hsdeviously . HsdC prA 20-pmole aliquot of the purified protein was incubated with 10 pmole of the dsDNA oligo for 15 min at 37 °C. The binding buffer contained 20 mM Tris-HCl (pH 7.5), 100 mM KCl, and 6% glycerol. Electrophoresis was performed using the PROTEAN II xi electrophoretic cell (Bio-Rad), TB buffer , and 6% acrylamide gel for 1 h at 450 V at 10 °C. After electrophoresis, the gel was stained with an ethidium bromide solution (3 μl of 1% EtBr per 400 ml of TB buffer) for 5 min and then visualized on a Typhoon scanner. The resulting images were analyzed using the ImageQuant software. All oligonucleotides used for EMSA are summarized in Table The HsdC binding constant was determined from the titration curve with a series of protein dilutions as described previously . BrieflyThe data on promoters’ position and activity was taken from our previous work . BrieflySearch for HsdC (C-protein) homologs was performed by NCBI blastp algorithm. Search for homologs of RM-systems proteins was performed using domain annotation from NCBI CDD database. Putative C-protein binding sites were identified by alignment of upstream regions of HsdC homologs in different species of Mollicutes.The raw data on transcription start sites (TSS’s) mapping was uploaded to NCBI SRA database under project id PRJNA325091.hsd operons (type I RM-system), while in other mycoplasmas, they are associated with type II DNA-methylases as well.Searching for HsdC homologs within Mollicutes identified a set of putative RM-system regulators. Homologs of C-proteins were distributed within different groups of Mollicutes, including Acholeplasmatales and Mycoplasmatales 3 sequence, although the spacer length varied by 1 nt in some occasions. However, at least two repeats featured a conserved 12 nt spacer. In mycoplasmas, we observed two major variants of the motif. One variant consists of a GTGTTA core sequence forming either direct repeats (GTGTTAN5)2 or inverted repeats. The latter variant was found in M. conjunctivae and in M. mobile. Another type of motif is completely different and consists of inverted repeats, GGACN5GTCC. This motif is similar to the well-characterized C-box of the AhdI RM-system AGTCCN2GGACT [Cross-species analysis of the respective promoters revealed putative HsdC binding motifs Fig. . To enhasepticum . The binsepticum , 11. Crosepticum . All stuM. gallisepticum S6, the C-protein homolog resides within one of the hsd operons coding for the type I RM-system. To identify the hsd operon promoter, we used a previously constructed whole-genome promoter map of M. gallisepticum S6 [hsdMSR genes. The first promoter encompasses direct repeats of the (GTGTTAN5)2 sequence, which partially overlap with the core promoter . The activity of the hsdC promoter  was app3)3, K = G or T [mraZ HsdC – PhsdC MraZ). MraZ protein is capable of binding to the HsdC motif with comparable strength as to its own (as a single octamer), while HsdC cannot bind to the MraZ motif. The MraZ-overexpressing strains obtained in our previous work [hsd operon in vivo (data not shown).The HsdC binding site resembles the core binding site of the MraZ transcriptional regulator  and the clpB chaperone gene. In both cases, the HsdC binding site overlapped with the -10 box of the promoter. To test their functionality, we performed EMSA as described above  where tove Fig. . In bothhsd operon is unclear. Extensively studied C-proteins of the AhdI [M. gallisepticum seems to act as a repressor of the whole operon of S, M and R subunits, and no other promoters were identified in the vicinity of the HsdC binding site. It is a question if HsdC functions only as auto-repressor of the hsd operon or is regulated on the post-translational level. In the first case it may produce burst-like expression rather than steady one.We identified a novel subfamily of transcriptional regulators of RM-systems, which are distributed within Mollicutes. The majority of them recognize motifs that are completely different from the known C-box in sequence and structure. There are three types of C-protein binding motifs in Mollicutes, and the most frequent ones consist of direct rather than inverted repeats. The recognition of direct rather than inverted repeats suggests an alternative protein dimerization mechanism as well. The exact role of HsdC in the control of the the AhdI  and PvuIthe AhdI  RM-systeM. gallisepticum S6 strain used in this work is currently the only strain of M. gallisepticum that features an RM-system with a transcriptional regulator. At the same time, all strains including S6 have another RM-system of type I that lacks a controller subunit. This observation as well as the duplication of the genomic region around the controlled RM-system indicates horizontal transfer of the whole operon , the binding site of HrcA repressor strains. This observation means that the ability to bind a novel regulator was acquired previously by chance. In the case of the clpB gene, the HsdC binding site was generated by an extended −10 box and a part of CIRCE, its dedicated regulator. This phenomenon may be considered as an event of exaptation [HsdC provides an example of an interesting evolutionary event: the acquisition and domestication of a foreign regulator. The promoter sequences of the aptation , where a12)3, and the others of mycoplasmas, GGACN5GTCC and (GTGTTAN5)2. The latter (the most frequent) was confirmed experimentally. The central TG dinucleotide was the most conserved and the most important for binding. Since the binding site of this type is relatively simple, the C-protein may bind other promoters, basically ones with a TRTG extension of the −10 box.Mollicutes feature homologs of the controller protein (C-protein) of RM-systems that regulate the transcription of both type I and type II RM-systems. In some cases, they form fusions with the corresponding methylase. There are three types of C-protein binding sites: one is characteristic of Acholeplasmas, (AACGAATN


IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, but etiology and pathogenesis continue to be poorly understood. Polymorphisms in the cytokine genes may play a role in the etiology and pathogenesis of IgAN. The incidence of different between diverse ethnic groups suggested important genetic influences on its pathogenesis.IL-1B and IL-6 gene using Sequenom Mass-ARRAY technology from 417 IgAN patients and 463 healthy controls of the Chinese Han population. We evaluated these SNPs associated with IgAN utilising the chi-square tests and genetic model analysis.We genotype 10 single nucleotide polymorphisms (SNPs) in IL-1B, IL-6 were involved in an increasingly risk of IgAN in allelic model analysis, respectively. The rs16944 in IL-1B and rs1800796 in IL-6 were associated with 1.23-fold  and 1.33-fold  increases in the risk of developing IgAN, respectively. There was only rs1800796 still correlated with IgAN in the allelic model after adjustment by age and gender and the Bonferroni correction. In addition, Haplotype Grs1800796A rs2069837G rs2069840 (P = 0.037) and G rs1800796A rs2069837C rs2069840 (P = 0.042) in IL-6were considered to be associated with increased IgAN risk.We identified that the minor alleles of rs16944 (“A”), rs1800796 (“G”) in IL-6, IL-1B genetic variants polymorphisms contributed to IgAN susceptibility in a Chinese Han population. Although we identified SNPs susceptibility, however, replication studies and functional research are required to confirm the genetic contribution in IgAN.This study verified the  Berger and Hinglais firstly described IgA nephropathy (IgAN) in 1968, and it is a relatively newly recognized disease . In the IL-6) contributed to the proliferation of cells [IL-6 encodes a cytokine that functions in inflammation and the maturation of B cells. And the encoded protein has turned out to be an endogenous pyrogen can induce fever in people who carried the infections or autoimmune diseases. The functioning of IL-6 is associated with a variety of disease states, such as diabetes mellitus and systemic juvenile rheumatoid arthritis.In IgAN patients, a previous study reported that Interleukin-6 . IL-1B coding for protein IL1-β is located in the 2q14 chromosome, has 7,153 kb and seven encoding regions. IL-1B plays a key role in the regulation of the immune responses and in the production of a variety of inflammatory mediators, together with other molecules [A study involved to patients with a progressive clinical course has reported that Increased xcretion . It has xcretion , 11. At ytokines . IL-1 exolecules .IL-1B and IL-6 polymorphisms and haplotypes on susceptibility to IgAN in a Chinese Han population from a case-control study. A total of 10 single nucleotide polymorphisms (SNPs) were studied in this association analysis in an attempt to identify markers that might guide intervention decisions and improve patient survival.In this study, we conducted an extensive association analysis to assess the roles of A total of 880 participants, including 417 IgAN patients and 463 controls, were analyzed in this study. Basic patient characteristics (gender and age) are listed in Table IL-1B and IL-6 gene were genotyped in the IgAN patients and the controls. Table P > 0.05), and two-sided Pearson chi-square tests were used to identify differences in allele frequency distributions between IgAN patients and controls.10 SNPs in the IL-1B, and rs1800796 in IL-6 were associated with 1.37-fold , 1.23-fold , and 1.33-fold  increases in the risk of developing IgAN, respectively. The SNP rs2853550 had a weak significant difference. Only one (rs1800796) of the three SNPs was still correlated with IgAN in the allelic model after Bonferroni correction.The rs2853550 and rs16944 in P-values were calculated using the chi-square test. The “A/G-A/A” genotype at the rs2853550 SNP increased the risk of IgAN in the dominant model , and “A/G” in the overdominant model with a P = 0.023 . The “G” allele in rs16944 decreased the IgAN risk in the log-additive model . However, these results were no statistical significance after adjustments for age and gender and the Bonferroni correction.And then, we determined the association between these 10 SNPs and IgAN risk using four genetic models Table ; P-valueP = 0.014) in the codominant model, the “C/G-G/G” genotype increased the risk of IgAN 1.45-fold  in the dominant model, and the “G” allele increased the risk 1.34-fold  in the log-additive model. In addition, after adjustment for age and gender, the “G” allele increased the IgAN risk 1.70-fold  in the codominant model, the “C/G-G/G” genotype increased the risk of IgAN more than 1.70-fold  in the dominant model, and the “G” allele increased the risk more than 1.50-fold  in the log-additive model. In the overdominant model, “C/G” increased the risk by 1.48-fold (P = 0.023) adjusted by gender and age. According to the adjusted by Bonferroni correction, mutations at rs1800796  were still associated with an increased IgAN risk.For the rs1800796 SNP, the “G” allele increased the risk 1.38-fold  analyses. LD was determined pairwise among all these 10 SNPs, the haplotype structure of the two genes were analyzed, two blocks  were found to increase risk of suffering from IgAN by more than 1.40-fold  and 1.90-fold , respectively. After adjusting for age and gender, the “Grs1800796A rs2069837G rs2069840”  and “Grs1800796Ars2069837Crs2069840”  haplotypes increased the IgAN risk 1.59- fold and 2.34-fold, respectively. Interestingly, for “Grs1800796Grs2069837Crs2069840”, there existed a statistically significant difference  after adjusting for age and gender.Finally, a haplotype-based association study was performed to examine associations between AN Table , and P-vThe onset of IgAN may be associated with infections in the upper respiratory tract. Genetic analysis of familial IgAN is the most promising approach to the identification of IgA disease , 15. ForIL-1B and IL-6 loci on susceptibility to IgAN. We identified the association between SNPs and the susceptibility of esophageal cancer in five genetic models , which equivalent of repeated multiple comparisons, increasing the chance of making type I errors. Meanwhile, we should perform the P correction with Bonferroni correction. We found that the rs2853550, rs16944 in IL-1B and rs1800796 in IL-6 genetic polymorphisms were correlated with an increased the IgAN risk in a northwestern Chinese Han patients population. To the best of our knowledge, this is the first case-control study to investigate this association. Persistent inflammation is linked with cancer development and progression [IL1-B, a key cytokine released from glial cells, is critically involved in the pathogenesis of chronic pain [IL1B can promote the invasiveness of breast cancer (BC) cells in vitro [IL1B induced osteoprotegerin secretion, independent of BC subtype and basal osteoprotegerin levels [IL1B gene. Many articles refer to associations in intronic regions and risk of diseases [IL1B in the additive model considering the “T” allele as risk showed association with the preeclamptic event [There is no doubt that there exist genetic components to the IgAN pathogenesis and IgAN clinical expression. In ourstudy, we systematically examined the impact of 10 SNPs in the nic pain , memory nic pain , via actnic pain . It has in vitro , 23. A sn levels . The poldiseases . The SNPic event . In our ic event . In thisIL-6 rs1800795 G>C polymorphisms presented an increased risk in dominant and recessive models, but they did not find that the IL-6 rs1800796 CC were correlated with an increased risk of coronary artery disease. A study reported that the rs1800796 TT genotype was associated with increased the susceptibility of cerebral thrombosis in the Chinese population [IL-6 gene and IgAN susceptibility in a Chinese population. Rs1800796 SNP was associated with IgAN risk, rs2069837 and rs2069840 showed no correlations with IgAN susceptibility. Further studies should involve some others regions and ethnic groups. None of previous study found that IL-6 haplotype was directly associated with IgAN risk. Our study detected that haplotype “Grs1800796Ars2069837Grs2069840” and “Grs1800796Ars2069837Crs2069840” are related with the IgAN increasing risk.GQ Sun et al.  found thpulation . Previoupulation . The frepulation . In our IL-1B and IL-6) polymorphisms and clinic pathological disease type were not evaluated in this study, Finally, both IgAN patients and controls were enrolled at the same hospital and therefore it may not be representative of the general population. Additional studies are needed to clarify the genetic mechanisms underlying IgAN by fine-mapping the susceptibility regions of the variants.Some limitations of the present study should be considered when interpreting the results. First, Several risk factors, such asgender, absence of macrohematuria, hypertension, severe proteinuria, smoking, and histopathologic changes, had been described to be associated with a poor outcome . But in IL-1B and IL-6 genetic polymorphisms are associated with an increasing risk of IgAN in a Chinese case-control population. This study offers new information on the relationship between two genes (IL-1B and IL-6) polymorphisms and IgAN susceptibility. Moreover, this study reveals the molecular markers associated with of IgAN susceptibility and could therefore be used as diagnostic and prognostic.In conclusion, we demonstrated that the All subjects were informed of the purpose of the study and the experimental procedures involved. The study protocol was approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University. We also obtained signed informed consent from all participants. The experimental protocol was implemented in accordance with the approved guidelines.From August 2012 to July 2016, 417 patients  were recruited to participates form Han population in the northwest China, all the cases were diagnosed as IgAN by renal biopsy and none-familialy IgAN cases. 463 healthy subjects  were recruited from routine healthy examinations in the same hospitals. All subjects were unrelated Chinese Han people living in Xi'an city or nearby. There are detail inclusion/exclusion criteria: patients with cancer, infection, secondary IgAN , other renal diseases and autoimmune diseases were excluded. The exclusion criteria for healthy subjects included the chronic disease, central nervous system-related disease, and conditions involving vital organs  and more aggressive metabolic and endocrinological disease.IL-1B and 3 SNPs in IL-6, and all of the 10 SNPs had minor allele frequencies (MAF) greater than 5%. Samples were centrifuged and stored at −80° until analysis. We extracted genomic DNA from peripheral blood samples using the GoldMag-Mini Whole Blood Genomic DNA Purification Kit  according to the manufacturer's protocol and measured DNA concentrations using a NanoDrop 2000. Sequenom MassARRAY Assay Design 3.0 Software was used to design primers for amplification and extension reactions [We examined 7 SNPs in eactions . SNP geneactions . Finallyeactions , 34.http://analysis.bio-x.cn/myAnalysis.php) to analyze linkage disequilibrium, haplotype construction, and genetic associations at polymorphism loci [Departure from Hardy-Weinberg Equilibrium (HWE) was assessed for the frequency of each SNP in genotype frequencies between the esophageal cancer and control groups were evaluated using the Chi-square test . Microsoism loci , 39.


The monitoring of [thiazole-14C]cefiderocol revealed the increased intracellular accumulation of cefiderocol in P. aeruginosa cells incubated under iron-depleted conditions compared with those incubated under iron-sufficient conditions. Cefiderocol was shown to have potent chelating activity with ferric iron, and extracellular iron was efficiently transported into P. aeruginosa cells in the presence of cefiderocol as well as siderophores, while enhanced transport of extracellular ferric iron was not observed when one of the hydroxyl groups of the catechol moiety of cefiderocol was replaced with a methoxy group. We conclude that cefiderocol forms a chelating complex with iron, which is actively transported into P. aeruginosa cells via iron transporters, resulting in potent antibacterial activity of cefiderocol against P. aeruginosa.Cefiderocol (S-649266) is a novel parenteral siderophore cephalosporin conjugated with a catechol moiety at the third-position side chain. The  Pseudomonas aeruginosa, resistance to β-lactam antibiotics is due to various resistance factors such as porin OprD deficiency, overproduction of efflux pumps such as MexA-MexB-OprM, overproduction of chromosomal AmpC β-lactamase, and acquisition of exogenous serine or metallo-β-lactamases such as Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron-borne metallo-β-lactamase (VIM), and imipenemase (IMP)  strains which are resistant to carbapenems, cephalosporins, aminoglycosides, and quinolones –3. In Ps–se (IMP) –13. SincP. aeruginosa, as well as iron uptake systems to scavenge for iron , a novel catechol-substituted siderophore cephalosporin, is structurally different from other recently developed siderophore-conjugated antibacterial agents and has shown potent aumannii –27. We h6, a noveumannii –. In this14C]cefiderocol were synthesized at the research laboratories of Shionogi & Co., Ltd. . The purity of [thiazole-14C]cefiderocol was 95.8%. Ceftazidime was purchased from U.S. Pharmacopeia   .P. aeruginosa PAO1 was kindly provided by Colin Manoil of the University of Washington .shington . P. aeruin vivo efficacy  chloride was added to ID-CAMHB. The iron concentrations in cation-adjusted Mueller-Hinton broth (CAMHB) and ID-CAMHB measured by inductively coupled plasma mass spectrometry  were approximately 0.2 mg/liter (3.6 μM) and 0.02 mg/liter (0.4 μM), respectively.MICs were determined by a broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) . For theefficacy –27. ID-CP. aeruginosa PAO1 was incubated at 37°C for 20 h in iron-depleted medium (ID-CAMHB) and iron-sufficient medium . The bacteria from each culture were washed and suspended in fresh media to achieve a bacterial suspension at an optical density at 625 nm (OD625) of 0.16 to 0.17. After the bacterial suspension in each medium was incubated at 37°C for 10 min, ammonium iron(III) citrate and [thiazole-14C]cefiderocol were simultaneously added to the medium at final concentrations of 21.2 μg/ml (approximately 66 μM) and 10 μg/ml (approximately 13 μM), respectively. One-milliliter samples from a total volume of 10 ml were taken at 0.5, 1, 3, 5, and 10 min and filtered through a 0.45-μm-pore-size nitrocellulose membrane filter (Millipore). The filtered samples were washed twice with ice-cold 0.05 M phosphate buffer (pH 7.0), and radioactivity was determined by a liquid scintillation counter . The amount of [thiazole-14C]cefiderocol taken up was calculated by the following equation: uptake amount = (radioactivity × 109)/(SPEC × MW) × (0.1/OD625), where radioactivity (expressed as the number of disintegrations per minute [dpm]) was determined by a liquid scintillation counter, SPEC is specific radioactivity (dpm/mg), and MW is molecular weight. Data represent averages of the results obtained by three independent experiments. A t test was conducted to determine the significant difference between results at each time point.P. aeruginosa, with ferric iron were determined by the color change of chrome azurol B (CAB) in the presence of cetyltrimethylammonium bromide (CTMA) . Data represent averages of the results obtained by three independent experiments. A t test was conducted to determine the significant difference between results at each concentration.Chelating activities of cefiderocol, cefiderocol catechol 3-methoxy, ceftazidime, and pyoverdine, which is the primary siderophore of e (CTMA) . A solutP. aeruginosa PAO1 was incubated in ID-CAMHB overnight at 35°C, and calcein-AM was added at a final concentration of 20 μM. At the same time, chloramphenicol was added at a concentration of 97.7 μg/ml to suppress the cell activity. The bacteria would not be killed by this step because the MIC of chloramphenicol against PAO1 is >512 μg/ml. After incubation for 120 min at room temperature under light-shielded conditions, the cells were washed with phosphate buffer, and bacterial density was adjusted to an OD600 of 5.0 in Vogel-Bonner minimal medium , a derivative of calcein, was used as a fluorescent indicator to determine the relative changes of the intracellular iron concentration . Once cal medium  supplemeP. aeruginosa PAO1 was diluted into fresh medium to yield an inoculum of approximately 1.0 × 108 CFU/ml. Aliquots of 0.1 ml were plated onto Muller-Hinton agar (MHA)  containing 4× or 10× the MICs of the compounds to be tested against the test strain and incubated at 37°C for 48 h. The number of resistant colonies was counted to determine the frequency of resistance.An overnight culture of P. aeruginosa PAO1, while the MIC of cefiderocol catechol 3-methoxy was 8 μg/ml. The treatment by cation binding resin resulted in a 4-fold decrease in the MIC of cefiderocol, and the MIC of cefiderocol was restored by the addition of 0.1 mg/liter of the iron, which was similar to the concentration of the iron in CAMHB. These results suggested that the concentration of the iron is a critical factor for determination of the antibacterial activity of cefiderocol. On the other hand, MICs of cefiderocol catechol 3-methoxy and ceftazidime did not change regardless of the iron concentrations in the medium. These results demonstrated the importance of the catechol moiety for the antibacterial activity of cefiderocol.MICs of cefiderocol, cefiderocol catechol 3-methoxy, and ceftazidime determined using CAMHB and ID-CAMHB supplemented with a range of ferric iron concentrations are shown in P. aeruginosa PAO1 grown in ID-CAMHB and CAMHB supplemented with iron were determined using radiolabeled cefiderocol, [thiazole-14C]cefiderocol . These results suggested that the enhanced uptake of cefiderocol into P. aeruginosa cells cultured in ID-CAMHB is associated with the increased activity of cefiderocol in ID-CAMHB.The uptake amounts of cefiderocol into iderocol . The uptP value < 0.05, by t test). The chelating activities were also significantly different between pyoverdine and ceftazidime and between pyoverdine and cefiderocol catechol 3-methoxy at the concentrations of 3, 10, 30, and 100 μM . These results showed that cefiderocol has chelating activity with ferric iron and that the catechol moiety is required for its chelating activity.The chelating activity of cefiderocol with ferric iron was determined by using CAB . CefiderP. aeruginosa PAO1 cells in the presence of cefiderocol was evaluated by calcein  the uptake of cefiderocol by P. aeruginosa is promoted in iron-depleted medium; (iii) the catechol moiety is required for the chelating activity of cefiderocol with iron and is also important for iron transport into P. aeruginosa. These experiments provide direct evidence that cefiderocol utilizes the iron transport system of P. aeruginosa to transport itself effectively by the binding of the catechol moiety with extracellular iron, mimicking the P. aeruginosa siderophore pyoverdine, which results in potent antibacterial activity. The antibacterial activity of cefiderocol as well as the uptake of [thiazole-14C]cefiderocol by P. aeruginosa cells was shown to be influenced by the iron concentration in the medium, which is consistent with the fact that the expression of the iron-regulated outer membrane proteins (IROMPs) is upregulated under iron-depleted conditions (The major findings in this study are as follows: (i) nditions , 35.in vitro antibacterial activity of cefiderocol is enhanced under iron-depleted conditions, and IROMPs are reported to be upregulated under iron-depleted conditions , consistent with earlier in vivo experiments using cefiderocol against P. aeruginosa (The nditions . The resnditions , 36, 37.sistance . Subsequruginosa , 27. Thein vitro activity of cefiderocol against P. aeruginosa were elucidated. Cefiderocol showed potent siderophore activity, and the catechol moiety on its side chain contributed to the chelating activity with ferric iron. Due to its catechol moiety, cefiderocol was transported more efficiently into bacterial cells under free-iron-depleted conditions than under free-iron-sufficient conditions, resulting in enhanced in vitro activity against P. aeruginosa.In this study, the underlying mechanisms responsible for the potent


Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti-P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptide’s structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa. Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients. Pseudomonas aeruginosa is the leading cause of lung infections and a primary cause of death in CF patients 2O as solvent, namely, (i) phosphate buffer ; (ii) phosphate buffer saline  and (iii) PBS with Na-EDTA . Peptide was dissolved at 2.0 mM in the presence of 20 mM large unilamellar vesicles (LUVs). The latter were prepared from a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and POPG by the extrusion method, as described in details elsewhere [3) was employed as reference for β-sheets [−1 spectral range, with 4 cm−1 resolution, by applying automatic atmosphere compensation and 32 scans. Background of the clean ATR crystal was acquired with the same parameters immediately before each measurement. Spectrum of buffers was also acquired in the same experimental conditions to be subtracted from samples’ spectra. Spectra were also recorded from the same samples as RF, obtained by depositing 10 μL of the dispersion on the ATR crystal and by gently evaporating the solvent under a stream of nitrogen. Rehydration was achieved by placing a small Petri dish over the film after deposition of several drops of D2O around the crystal. Spectra acquisition was started after one minute. Acquisition parameters were the same as for FH samples but 16 scans were acquired for the RF ones. The spectrum of the corresponding LUVs rehydrated film was acquired to be used for both medium and vesicles signals removal.Lin-SB056-1 was studied in three different media with Dlsewhere . The [POβ-sheets . All infP. aeruginosa strains was evaluated in sodium-phosphate buffer (SPB) 10 mM, pH 7.4 supplemented with 1% TSB (SPB/TSB) by the microdilution method. To this aim, bacterial strains were grown in TSB until exponential phase, washed by centrifugation and resuspended in SPB to reach a density of 1 × 107 CFU/mL. Bacteria, contained in a volume of 10 μL, were added to 90 μL of SPB/TSB containing the peptides at different concentrations (ranging from 0.78 to 12.5 μg/mL). To check for cell viability, samples containing bacteria suspended in SPB/TSB in the absence of the peptide were established. After an incubation at 37 °C with shaking for various time intervals , the samples were diluted 10-fold in TSB and plated on TSA for CFU counting. The MBC was defined as the minimal concentration of the peptide causing a reduction of at least 3-log in the number of CFUs after 90 min of incubation. In a set of experiments, peptide’s activity was assessed at 1.5 and 24 h in the presence of serum by adding heat inactivated pooled human serum, obtained from 6 healthy blood donors, to the assay mixture to reach a final serum concentration of 12.5%.The bactericidal activity of lin-SB056-1 against P. aeruginosa ATCC 27853 and the mucoid strain PaM01 were cultured overnight in TSB at 37 °C with agitation. After diluting bacterial suspensions 1:100 in TSB, aliquots of 100 μL were seeded into wells of flat-bottom polystyrene 96-well microtiter plates . Biofilms were left to form for 24 h (ATCC 27853 strain) or 7 days (PaM01 strain) at 37 °C in the absence of antimicrobial compounds. Previously it has been demonstrated that mucoid strains of P. aeruginosa need a supplement of calcium to produce appreciable amount of biofilm in vitro [570 absorbance was measured on a microplate reader .in vitro . In agrew/v); unsheared salmon sperm DNA (Sigma-Aldrich), 0.4% (w/v); NaCl, 0.5% (w/v); KCl, 0.2% (w/v); casamino acids (Oxoid), 0.5% (w/v); egg yolk emulsion . After each addition, the mixture was vigorously vortexed. The pH of the solution was adjusted at 6.8 with Tris-HCl 1 M, pH 8.5 [P. aeruginosa was grown overnight in LB broth, pelleted, washed with PBS, and resuspended in ASM at a density of 106 CFU/mL. A volume of 10 μL of the peptide (3.12 to 25 μg/mL) and/or 10 μL of EDTA (0.62 to 2.5 mM) were added to 80 μL of ASM containing bacteria. To check for cell viability, bacteria were suspended in ASM 80% alone. After an incubation at 37 °C with shaking for 90 min (or 24 h), the samples were diluted 10-fold in TSB and plated on TSA, to determine the number of viable cells.For ASM preparation, the components were dissolved into sterile water at the following concentrations: mucin (Sigma-Aldrich), 0.5%  conditions under microaerophilic conditions for different time intervals before assessing BLS formation. To this aim, 40 μL of each bacterial suspension was gently transferred onto a glass slide, covered with a coverslip and observed under light microscope . BLS content of a sample was determined dividing the sum of the score values by the number of microscope fields analyzed  and expressed in percent.p-value less than 0.05 was considered statistically significant. The data are means ± error standard of the mean (SEM) of three independent experiments.All statistical analyses were performed using one-way ANOVA followed by Tukey–Kramer post-hoc test. A P. aeruginosa. The combination of lin-SB056-1 with EDTA results in a potentiated antibiofilm effect against P. aeruginosa mature biofilms. The enhancement of the antibiofilm activity could be due to the destabilizing action of EDTA on the biofilm matrix as well as to a direct effect on biofilm-embedded cells. Importantly, in culture conditions mimicking the CF sputum, the combination lin-SB056-1/EDTA showed a significant bactericidal effect towards planktonic cells of P. aeruginosa and ability to almost completely inhibit the formation of BLS over a 72 h time-span. Future in vivo studies are planned to fully assess the therapeutic potential of the lin-SB056-1/EDTA combination in the therapy of P. aeruginosa infections in CF patients.Overall, the in vitro data obtained in this study demonstrated that the AMP lin-SB056-1 displays a very strong and rapid bactericidal activity against both mucoid and non-mucoid strains of


Furthermore, we observed decreased expression of miR-93 and miR-20a in MM-MSCs, while upregulation of miR-93/miR-20a decreased cellular senescence, as evidenced by the increased p21 expression. Importantly, we found that myeloma cells could induce the senescence of MSCs from healthy controls, as observed from the decreased expression of Dicer1 and miR-93/miR-20a and increased expression of p21. Overall, MM cells downregulate Dicer1 in MSCs, which leads to senescence; in turn, senescent MSCs promote MM cell growth, which most likely contributes to disease progression.Bone marrow mesenchymal stem cells (BMMSCs) facilitate the growth of multiple myeloma (MM) cells, but the underlying mechanisms remain unclear. This study demonstrates that the senescence of MM-MSCs significantly increased, as evidenced by a decrease in proliferation and increase in the number of cells positive for senescence-associated β-galactosidase activity. Senescent MM-MSCs displayed decreased differentiation potential and increased tumor-supporting capacity. Dicer1 knockdown in the MSCs of healthy controls promoted cellular senescence and tumor-supporting capacity, while decreasing the differentiation capacity. Dicer1 overexpression in MM-MSCs reversed the effects on differentiation and reduced cellular senescence. In addition, decreased expression of the microRNA-17 family was identified as a favorable element responsible for increasing senescence, with the expression of  This tissue is a complicated network of extracellular matrix and various cells2. Undifferentiated and pluripotent bone marrow mesenchymal stem cells (BMMSCs) are the principle members of the bone marrow microenvironment. BMMSCs promote MM cell proliferation via different factors, leading to increased tumor supporting capacity and the development of drug resistance. Evidence has shown that MSCs isolated from MM (MM-MSCs) exhibit different behavior from that exhibited by the MSCs from healthy controls (HC-MSCs), including differential gene expression, impaired cytokine release, and decreased osteogenic differentiation potential6. In MM, the interaction between BMMSCs and MM cells plays a pivotal role in MM development. MM cells attach to BMMSCs and release cytokines that promote MM cell viability, migration, and invasive ability, ultimately enhancing MM progression7. This interaction usually leads to complicated changes, including dysregulation of mRNA and miRNA expression and activation or deactivation of signaling pathways, but the underlying mechanisms remain unclear. For example, the development of a senescence-like state in BMMSCs, and thereby a modulated secretory profile, has been reported to worsen osteogenic differentiation potential11. Senescence is a cellular state corresponding with a decrease in proliferation ability and variation in the release of pro-inflammatory cytokines and growth factors12. Senescent BMMSCs exhibit an increased senescence-associated β-galactosidase activity  and irregular cell morphology14. Generally, cell cycle of the senescent cells is often arrested at the G0/G1 phase, in combination with the upregulation of different cell cycle inhibitors such as p2117. Increasing body of report suggests that microRNAs (miRNAs) take part in the regulation of MSC senescence19. Furthermore, they play an important part in the self-renewal and differentiation of MSCs. As previously indicated, MM-MSCs show a different miRNA profile to that of HC-MSCs, but the roles of these deregulated miRNAs in MM-MSCs are not clearly known21. The loss of Dicer1, an RNAse III endonuclease essential for miRNA biogenesis, and the ablation of mature miRNA result in the inhibition of cell proliferation and the induction of a premature senescence phenotype24. Our previous study showed that Dicer1 expression is reduced in MSCs from myelodysplastic syndrome patients (MDS) and that this down-regulation promotes cellular senescence and decreases the stem cell-supporting capacity of these cells17. However, the effect of the Dicer1 gene on the pathogenesis of MM has not been studied.Multiple myeloma (MM) is a malignancy characterized by plasma cell proliferation, initially in the bone marrow microenvironmentIn this study, we studied the senescent features of BMMSCs derived from MM patients and explored the biological function of Dicer1 in the senescence of MM-MSCs. Finally, we analyzed the decreased differentiation potential and the enhanced tumor-supporting capacity of MM-MSCs.p < 0.05, Fig. 0 peak, in accordance with hypodiploid apoptotic cells, was existed in the BMMSC cultures (<1%). Nevertheless, the percentage of MM-MSCs in G0/G1 phase was increased to 95.66 ± 1.26% , while the percentage of MM-MSCs in S phase was reduced to 2.64 ± 0.87%  when compared with that of HC-MSCs  when compared with non-senescent MM-MSCs .SA-β-gal was performed to identify the senescent MSCs. The number of SA-β-gal-positive cells was significantly elevated in MM-MSCs at each time point detection Fig. . From paoup Fig. . Moreove+) from MM patients also exhibited raised cell senescence, which was displayed by an obviously increased amount of SA-β-gal positive cells and increased p21 expression level, in comparison with the healthy control group. The collective data indicate that the proliferation capacity decreased and the senescence increased in MSCs from MM patients.In accordance with the above phenomena, primary MSCs  showed different disease progression compared to those with low β-gal staining MM-MSCs(NS-MM). Progression free-survival (PFS) was calculated from the date of the diagnosis to the date of progression. Patients with less than 4.4% SA-β-gal-positive cells at diagnosis, had significantly longer PFS  MSCs derived from MM patients and healthy controls. Results from RT-PCR and western blot exhibited that the expression of Dicer1 was declined in MM-MSCs  or with primary CD138SCs Fig. . The celWe then constructed a recombinant lentivirus to decrease the expression of Dicer1 to further study the biological effect of Dicer1 on MSC senescence. It is displayed in Fig. 1 phase and a decrease of those in the S phase without inducing apoptosis   Fig. . In the 01) Fig. .We also assessed the ability of MSCs differentiating into an adipogenic lineage treated with knockdown of Dicer1. On Day 21, Oil red-O-stained cultures revealed that excessive 91% of control MSCs could differentiate into adipocytes, exhibiting plenty of cellular lipid droplets. On the contrary, the cellular lipid amount in Dicer1-KD MSCs was rare, consisting of small droplets , negative MSCs (transfected with control lentiviruses), Dicer1-KD MSCs (shRNA), and MM-MSCs for 24 h. Cell cycle behavior of NCI-H929 cells was identified by Click-iT Edu Flow Cytometry Assay. Fluorescence-activated cell analysis plots from one representative experiment showing the cell cycle plots for monocultured MM cell lines (NCI-H929) or with MSCs as indicated. The location of cells in different phases of cell cycle is shown in the left histogram, and the percentage of cells in S is obtained for each condition Fig. . The res+cells were cultured in complete medium with 5 nM Bortezomib for 48 h, approximately 33.4% apoptotic cells (early and late) were observed. However, in the presence of MSC-Dicer1-shRNA and MM-MSC, the proportion of apoptotic cells significantly decreased to 9.8% and 10.1%, respectively , DKK-1 , and IL-6 (interleukin-6) compared with control MSCs. However, the level of SDF-1 was significantly under-expressed in MM-MSCs compared to HC-MSCs. Cytokines were evaluated by real-time RT-PCR and ELISA.  or green fluorescent protein (AD-GFP). The mRNA and protein expressions of Dicer1 was significantly elevated in AD-Dicer1 MSCs Fig.  levels. p21 levels after 48 h transfection was also detected. In three analyzed samples of MM-MSCs, the overexpression led to decreased p21 expression . It is clear that the amount of SA-β-gal positive cells was significantly higher in MM-MSCs than HC-MSCs. Our findings are in agreement with previous studies, but these studies did not detail the mechanisms and functional changes associated with senescence in the pathogenesis of MM.27. In the current study, knockdown of Dicer1 in HC-MSCs decreased the osteogenic differentiation of MSCs. Moreover, MM-MSCs exhibited an increased tumor-supporting capacity. Thus, the senile MSCs could be a promoting element in favoring myeloma cell growth of patients with MM. Furthermore, knockdown of Dicer1 in HC-MSCs promoted cellular senescence and tumor-supporting capacities, while decreasing the differentiation of MSCs. We confirmed that MM-MSCs and Dicer1-KD MSCs increased the proportions of S phase myeloma cells. In accordance with our findings, some studies showed that MM-MSCs displayed tumor-supporting capability compared with healthy controls. However, some of the previous reports demonstrated that MSCs possibly have inhibition capability. For example, Ramasamy et al. reported that MSCs act directly on tumor cells, suppressing proliferation and inducing apoptosis in tumor cells28. Whether MSCs promote or inhibit myeloma cell growth remains controversial. The diversities in these findings are probably due to the different origin of MSCs, cellular populations and experimental protocols in the respective studies.The characteristics of senescent MSCs include changes in cellular functions. We demonstrated that senescent MM-MSCs displayed decreasing differentiation capability and increased tumor supporting ability. The depressed osteogenic and adipogenic potentials in senescent MM-MSCs have been verified in vitro by quantitative and continuous detection. In particular, some cell surface molecules and subsequent downstream signaling pathways take part in the regulation of MM-related bone destroying process, in which the balance of bone resorption and formation are no longer acts as a result of the increasing activity of osteoclasts, but rather the osteoblast activity is decreased, leading to an uncoupled or severely imbalanced bone remodeling process. In addition, our previous study showed that MM patients exhibit an impairment of osteogenic differentiation of BMMSCs as compared with healthy controls19. It is reported that Dicer1 is an RNAse III endonuclease responsible for microRNA biogenesis22. Furthermore, our preliminary findings showed that the mRNA and protein levels of Dicer1 in MSCs isolated from MDS patients was significantly decreased compared with MSCs from health controls17. On the basis of these findings, we evaluated Dicer1 expression in MM-MSCs. It was demonstrated that the expression of Dicer1 decreased in primary and expanded MM-MSCs. In the current study, we investigated the effects of a lower expression of Dicer1 in MM-MSCs by generating a stable knockdown model. Our study is mainly concerned with the biological function of Dicer1 in cell senescence, differentiation and tumor support of Dicer1-KD MSC expedited cellar senescence, which manifested as flattened cell morphology, increased SA-β-gal positive incidence and a decreased percentage of cells in S phase. Experiments involving Dicer1 overexpression further verified these results. Upregulation of Dicer1 expression in MM-MSC reversed cellular senescence and promoted differentiation. Under-expression of Dicer1 interferes with the biogenesis of miRNA, which is relevant to cellular senescence in endothelial cells. However, the role of miRNA in the senescence of BMMSCs remains ambiguous. Senescent cells generally exhibit increased expression of cell cycle inhibiting factor such as p53/p21, which could be adjusted by miRNA31. In our current study, decreased expression of the miR-17 family  was determined to be a critical factor responsible for elevated p21 expression in Dicer1-KD MSC. Actually, miRNAs in the miR-17 family have been identified as regulators of cell cycle through targeting p21 in many other studies34. It was demonstrated that a lower of expression of the miR-17 family could give rise to the Dicer1-KD-induced cell senescence in MSCs in our present and previous studies. Lately, substantial miRNA expression was reported to be downregulated in MM-MSCs, demonstrating that reduced miRNA expression probably be associated with the pathogenic mechanism of MM. Our result showed that expression of miR-93 and miR-20a remarkably decreased in MM-MSCs. Moreover, overexpression of miR-93/ miR-20a reversed cellular senescence in MM-MSCs through targeting p21. It was confirmed that miRNA showed a critical effect on increasing cell senescence of MM-MSCs.In recent years, miRNAs, a pivotal group of moderating factors, have been deemed to be prominent regulators of cellular senescence37. Our study reported that the reduction of miRNA processing induced by Dicer1-KD was effective to immensely decrease the differentiation ability of MSCs, which indicated that exact level of mature miRNAs is important for MSC osteoblast differentiation. Most importantly, it provides a new perspective for the study of bone lesions in MM.Dicer1-KD MSCs displayed diminished osteogenic differentiation potential, characterized by the reduction in the expression of osteoblastic maker genes, calcium deposits and ALP activity, which was consistent with previous studies. Furthermore, upregulation of Dicer1 increased the impaired osteoblastic differentiation capability of MM-MSCs. Recently, many studies demonstrated that osteolytic bone lesions of MM patients resulted from the abnormal osteoblastic function in patientsThe senescent MSCs not only exhibited an impaired differentiation potential but also an enhancement in their support of tumor growth. So far, knowledge concerning the effect of MSC senescence in tumor growth support is restricted. In our results, in addition to the classic features of cell senescence, Dicer1-KD MSCs exhibited a significant increase in their capacity to support myeloma cells. Furthermore, BMMSCs derived from patients with MM were found to deliver the cytokine IL-6, which is known to promote tumor formation and progression. The tumor microenvironment-derived GDF15 was also able to significantly increase cell survival in primary MM cells.In this study, we explored the feature and molecular mechanism of MSCs senescence in MM patients. Dicer1 plays a crucial role in the senescent process of MM-MSCs by regulating miRNA. Senescent MM-MSCs have a decreased differentiation potential and an increased tumor-supporting capacity. Therefore, much attention should be paid to the therapeutic exploitation of MSCs in bone lesion diseases and tumor progression.n = 46) were enrolled between January 2015 and June 2017 in this study . The bone marrow was extracted for experiments before the treatment start. Eighteen healthy controls with the median age of 66 years  were analyzed in our study and they were matched for gender and age.A total of forty-six patients with newly diagnosed MM , supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 mg/mL streptomycin.The proliferation of BMMSCs was detected by using CCK-8  and by measuring the cell doubling time from P1 to P5. To evaluate the clonogenic potential of BMMSCs, colony-forming unit fibroblast (CFU-F) at P1, P3, and P5 were counted (colonies were stained with Giemsa).2 at 37 °C. NCI-H929 were co-cultured with MSCs (HC-MSCs or MM-MSC) for 24 h. We used a trans well inserts with 1-μm pores. MM cell lines or MM primary cells were cultured in the upper chamber of the inserts.  in culture medium for 45 min. EdU was detected with Alexa Fluor 647-azide with the use of a Click-iT EdU Flow Cytometry Assay Kit (Invitrogen). Flow cytometry was performed with a FACS Calibur flow cytometer  and Cell Quest software.The human multiple myeloma cell lines NCI-H929 were purchased from the American Type Culture Collection . The cells were cultured in RPMI 1640 (Gibco) medium supplemented with 10% fetal bovine serum (Gibco),100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% COBMMSCs were plated into 6 well culture-plates and maintained until 80% confluence. The cells were incubated with SA-β-gal staining solution  according to the manufacturer's instructions. Under inverted microscope, the number of SA-β-gal positive cells (blue cells) were calculated as senescent cells.Cells were fixed by precooled ethanol for at least 24 h and stained with the PI/RNase Staining Solution kit  according to the manufacturer’s directions. The cell cycle analysis was evaluated by flow cytometry and MultiCycle software.Apoptosis of MM cells was induced by Bortezomib . The quantification of apoptotic cells was performed by using the AnnexinV-FITC Apoptosis Detection Kit  according to the manufacturer’s instructions. The analyses were performed on the FACScan flow cytometer.40.Total RNA was isolated using the RNeasy Mini Kit  according to the manufacturer’s instructions. For mRNA detection, reverse transcription of RNA was performed using the ReverTra Ace qPCR RT Kit , and real-time PCR (RT-PCR) was carried out using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) . The primers are listed in 41.The cellular lysate was fractionated by 12% SDS-PAGE and electroblotted onto PVDF membranes. Membranes were incubated with primary antibodies including anti-Dicer1 , anti-p21  and anti-βactin . Secondary goat anti-rabbit antibodies labeled with horseradish peroxidase (Amersham Biosciences) were used. The process was detailed in our previous study17.The HC-MSCs were transfected with shDicer1-eGFP vectors (shRNA group) and negative vector. And detailed information about cell transductions is provided in our previous articles41. The MM-MSCs were planted in 6-well plates and cultivated for 24 h. The cells were transfected with 20 μl adenoviral vectors and cultured for 12 h and then the medium was changed. The cells were cultured for another 36 h and harvested. The transfection was performed according to the manufacturer's protocol.The process of the construction of adenoviral vectors for expression of Dicer1 was detailed previouslyMM-MSC were transfected with human miR-20a, miR-93 and scrambled control lentivirus  according to the manufacturer’s protocol.t test, one-way analysis of variance (ANOVA) was used to assess multiple pairwise comparisons p < 0.05 was considered statistically significant. Estimation of PFS was performed using the method of Kaplan and Meier.The data were presented as mean ± SD. All statistical analyses were performed using the SPSS 17.0 System. Comparison of mRNA levels between healthy controls and different MM subtypes was using a Student’s Supplementary TableSupplementary legendFigure S1Figure S2Figure S3Figure S4


H. pylori infection. The area under the ROC curve for HULC was up to 0.888, which was higher than that for CEA (0.694) and CA72-4 (0.514). Follow-up detection and Kaplan-Meier curve analysis revealed HULC is a good predictor of GC prognosis. Our present study indicates that circulating HULC may represent a novel serum tumor marker for early diagnosis and monitoring progression and prognosis of GC.Long non-coding RNAs (lncRNAs) have recently emerged as vital players in tumor biology with potential value in cancer diagnosis, prognosis, and therapeutics. The lncRNA HULC (highly up-regulated in liver cancer) is increased in many malignancies, yet its serum expression profile and clinical value in gastric cancer (GC) patients remain unclear. Quantitative real-time polymerase chain reaction (RT-qPCR) for large-scale analysis of the serum expression of HULC in GC patients reliably detected circulating HULC and revealed that it is upregulated in GC patients. A high serum HULC level correlated with tumor size, lymph node metastasis, distant metastasis, tumor-node-metastasis stage, and  Gastric cancer (GC) is one of the most common malignancies and the second leading cause of cancer deaths worldwide . It contAt present, serum carcinoembryonic antigen (CEA) and carbohydrate antigen 72-4 (CA72-4) are the most widely used tumor markers for GC. However, their clinical use is limited by low sensitivity and specificity . Easier,Circulating nucleic acids (CNAs), such as circulating cell-free DNA (cf-DNA) and microRNA (miRNA), are extracellular nucleic acids found in serum, plasma, or other body fluids. Many studies , 7 have LncRNAs are transcripts longer than 200 nucleotides with little or no protein coding function. They are considered a new class of regulatory ncRNAs. Mounting evidence shows that lncRNAs play important roles in a large range of biological processes and diseases, especially in cancer biology . CompareHULC (highly up-regulated in liver cancer) is overexpressed in liver cancer  and can 2 of the HULC standard curve was 0.9949, and the regression equation was y = −3.047x+21.95, indicating that qPCR could be used to detect different concentrations of serum HULC , glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (β2M), and ribosomal and 18S ribosomal RNA (18SrRNA) for comparison by RT-qPCR in 30 samples . Because glyceraldehyde-3-phosphate dehydrogenase (GAPDH) had good linearity and reproducibility without being affected by age, sex, and pathologic grade Figure , it was C Figure . The intC Figure . In addiC Figure .Many laboratories use pre-amplification to improve sensitivity . In the The origin of circulating lncRNA remains a mystery at present. In our study, we found no significant difference in the expression of HULC in four GC cell lines AGS, SGC-7901, MGC-803, and MKN-45 as compared with GES-1 Figure . InteresTo further validate the interaction between GC and HULC, RT-qPCR analysis was performed to determine the expression level of HULC in 100 primary GC patients, 30 polyp patients, and 110 normal controls. The results showed that serum HULC was highly expressed in GC patients, as compared with the polyps and normal groups (P < 0.001) Figure . The meavs. 0.694  for CEA and 0.514 (95% CI: 0.433–0.595: P = 0.737) for CA72-4  curves were constructed on data from all subjects, including 100 GC patients and 110 healthy controls. Serum HULC effectively differentiated primary GC patients from normal controls with an area under the ROC curves (AUC) of 0.888  4 Figure . At the H. pylori infection (P = 0.046). We did not find any correlation between serum HULC expression level and other clinicopathological parameters, such as gender, age, and serum CEA level.The clinicopathological features of the 100 GC patients are summarized in Table Next, we investigated the expression of HULC in 100 primary GC patients, 62 surgical patients, and 11 recurrent patients. The level of serum HULC was significantly lower in the surgical patients than in the primary GC patients and recurrent patients (P < 0.001) Figure . Pre- anThe prognostic value of HULC expression was investigated using the Kaplan-Meier method. As shown in Figure In recent years, genome-wide transcriptional studies have indicated that only approximately 2% of the human genome produces biologically meaningful RNA transcripts. A much larger proportion of the genome is transcribed into non-coding RNAs (ncRNAs) , 80% of in vivo and in vitro. HULC has obvious induced effects on other digestive system carcinomas, such as GC and pancreatic cancer [LncRNA HULC, a liver cancer-associated lncRNA mapped to chromosome 6p24.3 , was fouc cancer , 32. ZhaH. pylori (HP)-positive group was higher than that in the HP-negative group (P = 0.046). Large-scale epidemiological studies have confirmed that HP is a strong risk factor for both GC development and progression. These findings indicate that HULC may be involved in the pathogenesis of GC. Furthermore, ROC to evaluate the diagnostic utility of HULC indicates that serum HULC provides a more powerful differential ability than CEA and CA72-4, suggesting that serum HULC could serve as a promising tumor marker for GC diagnosis. Finally, to investigate the prognostic role of HULC on GC, we performed Kaplan-Meier analysis indicating that HULC could be a marker for outcomes prediction.Our study is the first to quantify HULC in human serum by using conventional RT-qPCR with good linearity, specificity and reproducibility. We demonstrated that serum HULC expression is significantly higher in primary GC patients than in normal controls and individuals with gastric polyps, while there was no significant difference between the latter two groups. We also found that HULC levels underwent dynamic changes as the disease progressed from a precancerous lesion to cancer. Moreover, serum HULC was significantly decreased in the post-treatment patients to a level that was similar to that in healthy individuals. In addition, the expression of serum HULC quickly rebounded in patients with a collapsed condition even after surgical treatment. Our results show that the level of HULC expression was associated with adverse prognostic factors, and intriguingly we found that the serum HULC level in the In conclusion, our results suggest that increased serum HULC could be an ideal tumor biomarker for GC detection. Simultaneously, serum HULC has potential clinical value for forecasting prognosis and predicting the therapeutic efficacy for GC.Recent studies have demonstrated that CNAs are detectable in plasma and serum of cancer patients with dysregulation panels , 33, 34.We observed that serum HULC is surprisingly stable. The underlying mechanism of circulating lncRNAs’ stability in the presence of large numbers of RNases remains unclear. As our study was a preliminary report on the clinical value of circulating HULC in GC, further studies should focus on large-scale sample collection, long-term follow-up, and in-depth functional investigation in order to explore the potential value of HULC in GC.Serum samples from 173 GC patients, 30 patients with gastric polyps, 30 patients with high atypical hyperplasia or intestinal metaplasia, and 110 age-matched healthy controls  were collected. In addition, additional 60 normal samples were recruited to mix together as control in each qPCR experiment. The 173 GC patients consisted of 100 patients with primary GC who had not received any treatment before surgery, 62 surgical patients, and 11 recurrent patients. Forty of the 62 GC patients who received surgery were followed up for one month. All blood samples of the patients with GC, precancerous lesions, and polyps were obtained from the Departments of General Surgery and Gastroenterology at the Affiliated Hospital of Nantong University  between July 2012 and July 2014. The 170 normal controls were selected from volunteer blood donors of Nantong Blood Center who had no history of autoimmune disease, tissue injury, or trauma at the time of examination. The diagnosis of all 173 GC patients was confirmed by pathology. Histologic grading was performed according to the tumor-node-metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC) Clinical Practice Guidelines for Oncology (2011).All samples from 231 males and 161 females with a mean age of 60 years (range 26-85) were anonymous with written informed consent. The study was approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University.Blood samples were collected in separating gel vacuum collection tubes and centrifuged at 3000×g for 10 min. The upper-layer supernatant was immediately stored in an RNase-free Eppendorf tube at −80°C until use.TM, IMPLEN, GER). Complementary DNA was extracted from 10 μg total RNA using the RevertAid First Strand cDNA Synthesis Kit . The mix was incubated at 42°C for 60 min and 70°C for 5 min. The reverse transcription products were stored at −20°C until use for RT-qPCR.Serum RNA was extracted from 400 μl serum using Ambion mirVana PARIS kit  according to the manufacturer's protocol. The RNA concentration and purity were measured with a spectrophotometer (NanoPhotometer−ΔΔCT). The primers used were as follows: HULC-forward 5′-TCATGATGGAATTGGAGCCTT-3′, HULC-reverse 5′-CTCTTCCTGGCTTGCAGATTG-3′; GAPDH-forward 5′-CCCTTCATTGACCTCAACTA-3′, GAPDH-reverse, 5′-TGGAAGATGGTGATGGGATT -3′.RT-qPCR was performed on Applied Biosystems 7500  with 3.0 μl cDNA  and 10.0 μl FastStart Universal SYBR Green Master Mix . The PCR cycling program consisted of incubation for enzyme activation at 95°C for 10 min, followed by denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and then extension at 72°C for 30 s, for a total of 45 cycles. The expression levels of HULC were normalized to the internal control GAPDH to obtain the relative threshold cycle (ΔCT) and mixed normal sera were used as control to calculate relative expression levels  and gastric cancer cell lines  were purchased from the Chinese Academy of Sciences Cell Bank . Cells were cultured in 1640 medium  with 10% fetal bovine serum (FBS) and 1% antibiotics  at 37°C in a humidified incubator under 5% COSerum CEA and CA72-4 were measured with ARCHITECT I2000 SR . The cutoff value used to make a distinction between positive and negative results was 5 ng/ml for CEA and 10 U/ml for CA72-4.Statistical analysis was performed using the SPSS software package version 20.0 . Statistical significance was tested by a Mann-Whitney unpaired test analysis of variance or a Chi-square test as appropriate. P < 0.05 was considered statistically significant. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were used to assess the diagnostic value of using HULC and CEA for GC. The figures were partially drawn by GraphPad Prism 5.0 software .


Its luminescent properties have been intensively investigated for samples, in the form of bulk, thin film, or nanostructure, prepared by various methods and doped with different impurities. In this paper, we first review briefly the recent progress in this field. Then a comprehensive summary of the research carried out in our laboratory on ZnO preparation and its luminescent properties, will be presented, in which the involved samples include ZnO films and nanorods prepared with different methods and doped with n-type or p-type impurities. The results of ZnO based LEDs will also be discussed.Zinc oxide (ZnO) is a potential candidate material for optoelectronic applications, especially for blue to ultraviolet light emitting devices, due to its fundamental advantages, such as direct wide band gap of 3.37 eV, large exciton binding energy of 60 meV, and high optical gain of 320 cm Intensive research efforts have focused on ZnO and related devices for many decades. The quality of ZnO in the forms of both bulk and thin film has been improved substantially. The nanostructured ZnO materials have attracted great attention and various dimensions and shapes have been prepared. On the other hand, the luminescent properties of the ZnO related materials have been extensively investigated. The ZnO based LEDs of various device structures have been demonstrated. In addition, the exploration of new preparation methods is still in progress. Zinc oxide (ZnO) based materials are potential candidates for optoelectronic applications, especially for blue to ultraviolet light emitting devices, due to its fundamental advantages, such as direct wide band gap 3.37 eV, large exciton binding energy 60 meV, and high optical gain 320 cmIn terms of the ZnO related investigations, we can say that great progress has been achieved, but there are still some obstacles to be overcome for realizing its wide optoelectronic applications, which remains a hot research direction. Large numbers of researchers are steadily working to explore this potential and promising field, and thousands of research papers are published each year. It is noted that various aspects of the investigation have been reviewed, such as in the comprehensive review papers ,2. In thThis paper is arranged as follows. Growth of bulk ZnO crystals is mainly carried out by hydrothermal ,4,5,6, vRf sputtering is one of the most popular growth techniques for ZnO thin films. Although the earlier sputtered materials were polycrystalline or even amorphous, some research  has repo−3 Ωcm can be obtained without post annealing  was observed from the annealed Al–N codoped ZnO film diffraction pattern. The appearance of the zinc nitride phase was believed to be the nitrification reaction of the excess Zn and N atoms in the codoped films, indicating the excitation of the N ions after thermal annealing. P-type conductive behavior of AlN codoped ZnO was obtained after an additive post-annealing treatment at temperatures ranging from 400-600 °C under nitrogen ambient for 30 min, which will be discussed in We have developed a vapor cooling condensation method , with whi-ZnO film was deposited on the substrate cooled by liquid nitrogen. An electron concentration of 7.6 × 1015 cm−3 and mobility of 2.1 cm2/Vs of the deposited i-ZnO film at room temperature were obtained. The room temperature PL spectrum of the resultant i-ZnO film is shown in n-ZnO films deposition with pure ZnO and In as source materials. The electron concentration and mobility of the deposited ZnO:In films were 1.7 × 1020 cm−3 and 3.7 cm2/Vs, respectively. The PL spectrum of the n-ZnO:In films is also presented in By heating the tungsten boat loaded with 0.85 g of ZnO powder, a 300 nm thick 1–xMgxO  films annealed at 450 °C are shown in All these suppositions were confirmed by the results of XRD, low temperature PL spectra (LTPL) and time-resolved PL spectra (TRPL) measurements. 2 on O substitution sites (N2)O simultaneously existed in samples D and E, which resulted in the larger lattice constants as identified by the XRD experimental results (not shown here). This was consistent with the fact that samples D and E were deposited under higher N2/(N2 + Ar) flow ratios. Furthermore, the fact that the intensity of 2.35 eV emission band in samples D and E is higher than that in sample C implies more O vacancies existed in samples D and E, which can be tentatively interpreted as the result of compensation for the existence of more (N2)O in the film. Because both O vacancies and (N2)O serve as donors in ZnO film, N acceptors formed in samples D and E were partly compensated by them and resulted in a lower hole concentration as seen in 2 /(N2+Ar) = 4%]) annealed at 400, 450, and 500 °C, respectively. The broad blue-green emission band around 2.4–2.8 eV was originated from the deep level caused by the dopant-induced defects. The other broad deep level emission at 2.15 eV was regarded as the oxygen-related emission. As shown in O acceptors, which interprets the stable p-type behavior for the sample in group C annealed at 450 °C. The degradation of the p-type behavior for samples annealed at higher temperatures was attributed to the dissociation of Zn–N bonds and Zn–O bonds. Compared to the case of the sample C, more Np-type ZnO films can be obtained by co-sputtering of ZnO and AlN under an adequate N2/(N2 + Ar) flow ratio of 4% and post-annealing at 450 °C.The results discussed above lead us to the conclusion that high quality et al. [et al. [2O as a dopant source. Low-temperature growth routes for ZnO nanorods have also been reported [etc.It is well known that nanometer semiconductor may have superior optical and electrical properties than bulk crystals owing to quantum confinement effects. Great efforts have been devoted to the nano-science and technology in the world. Nanostructured ZnO is also a hot topic in the research community, especially one-dimentional (1D) ZnO nanostructures. Large amount of research reports on 1D nanostructured ZnO of different shapes have been published and comprehensively reviewed . Here weet al.  have use [et al.  reportedreported ,125. 2D In our lab, several deposition methods have been used to grow nanostructured ZnO layers, such as low pressure vapor phase transport process , carboth2, around 1.0%, into flowing Ar during growth significantly enhanced the growth rate and UV emission of ZnO nanodisks, while the green emission was significantly suppressed when the oxygen concentration was increased from 0.5 to 1%. The latter can be attributed to the reduction of oxygen vacancies and surface defects in ZnO nanodisks. It is noticed that when the oxygen concentration was increased to 10%, the intensity of UV emission was considerably reduced. It is conceived that at a higher oxygen partial pressure many Zn vacancies may form in the ZnO nanodisks, resulting in suppression of UV emission.We fabricated In-doped ZnO nanodisks  by carboZnO nanorod arrays with different densities and sizes were also fabricated by using a chemical vapor transport process in our lab . The souWe also fabricated vertically well-aligned ZnO nanowires by spin-on-glass technology on ZnO:Ga/glass templates , which het al. studied theoretically the optical properties of zero-dimensional ZnO nanostructures or quantum dots (QDs). They found that depending on the fabrication technique and ZnO QD surface quality, the origin of UV photoluminescence in ZnO QDs with diameters from 2–6 nm was due to the recombination of either confined excitons or surface-bound ionized acceptor–exciton complexes. In the latter case the Stokes shift of about 100–200 meV was observed in the photoluminescence spectrum. They also found that the radiative lifetime of the confined exciton and that of the ionized donor–exciton complex were almost the same. The lifetimes in both cases decreased with QD size and were about an order of magnitude less (for QD of radius R~2 nm) than the bulk exciton lifetime. For the QD with diameter 5 nm the lifetime of 38 ps was obtained, in agreement with the conclusion of Bahnemann et al. [R~2 nm, the lifetime was about two orders of magnitude larger than the bulk exciton lifetime. Thereby they proposed to use the exciton radiative lifetime as a probe of the exciton localization. Laser action from nanostructured ZnO is another interesting topic that has been studied widely. As an example, X.H. Han et al. [In addition to the preparation of the nanostrutured ZnO, their optical properties are another important issue to be exploited. Generally the optical properties of nanostructured semiconductors depend strongly on their size and shape , which in et al. . This isn et al.  demonstrD0X at 3.359 eV), the 1-LO(71 meV), 2-LO, 3-LO, and 4-LO phonon replicas of donor acceptor pair (DAP at 3.315 eV ) transition, and the peaks at high energy shoulder  further indicates (besides the FE-SEM image and the XRD pattern ) that ZnO nanowires are of high optical quality.Short wavelength luminescence of nanostructured ZnO is of great interest due to its potential optoelectronic application. Because of its fundamental importance we studied exciton related interaction in the ZnO nanostructure. We successfully fabricated high quality ZnO nanowires with a diameter of about 70 nm and 1000 nm in length on Si (100) substrates by a low pressure vapor phase transport process , and thep-ZnO materials, more research efforts have been devoted to the exploration of various ZnO-based short wavelength light-emitting diodes. The progress in the aspect goes ahead continuously and new improvements have been demonstrated in recent years, as summarized in a recent publication [Due to the progress in preparation of ZnO materials, especially lication . p-n homojunction LEDs is still an attractive research topic. The progress achieved in this direction is closely related to the improvement in preparation of p-type ZnO materials. It is not difficult to list a few newly reported examples of ZnO p-n junction LEDs, where p-ZnO was obtained by using different dopants, such as NO [2O [2-O2 [p-n junction LEDs emitted blue-violet electroluminescence under forward bias at low temperature, which was attributed to donor-acceptor pair recombination in p-ZnO. Some works also reported the emission from deep defect levels. It is noted that device performance improved, but the EL was still quenched substantially at room temperature. This can be ascribed to the imperfection of the p-ZnO. Firstly, the exploration of ZnO ch as NO , N2O [13s NO [2O , N2-O2 [2O [2-O2 , N-Al [12O [2-O2 , N-In [12O [2-O2 , and Sb 2O [2-O2 . Variousp-ZnO is still not available, many efforts have continuously focused on an alternative type of device structures, the n-ZnO based heterojunction , in whiet al. [n-ZnO/ p-GaN:Mg heterojunction LED on c-Al2O3 substrates using pulsed laser deposition for the ZnO and metal organic chemical vapor deposition for the GaN:Mg. Room temperature electroluminescence of the LED showed an emission peaked at 375 nm, the same as the photoluminescence of the ZnO layer, indicative of a near band edge excitonic emission. Over the injection current range from 500–875 mA the light-current (L-I) characteristic of spectral intensity versus current appears fairly linear.Rogers et al.  fabricatet al. [n+-GaN/sapphire substrates using a radio frequency magnetron sputtering system. Near band edge emission of ZnO peaking at 370 nm was achieved at room temperature when the devices were under sufficient forward bias. It is also demonstrated that the device performance was improved by inserting an electron blocking i-Mgx1−ZnxO (0 < x < 1) layer.Long et al.  reportedet al. [2O3 substrates. The white EL was produced by superposition of two strongest emission lines, a narrow blue line at 440 nm and a broad yellow band around 550 nm, which was attributed to the high density of structural defects at the n-ZnO/p-GaN interface.Titkov, Delimova et al.  demonstret al. [n-ZnO/ (InGaN/GaN) multi-quantum-wells/p-GaN, which was grown on semi-insulating AlN/sapphire using pulsed laser deposition for the n-ZnO and metal organic chemical vapor deposition for the other layers. LEDs showed a turn-on voltage of 2.5 V and a room temperature EL centered at 510 nm.Bayram et al.  reportedet al. [2 and SiNx on ZnO-based heterojunction p-i-n LEDs.Besides the development of various device structures, the surface passivation was also used to improve the performance of the devices. For example, Yu-Lin Wang et al.  investigp-ZnO has also been used to construct hetrostructure LEDs in combination with other materials. However, many of the devices yield deep-level related emissions with none or small UV emissions. Mandalapu et al. [p-ZnO/n-Si heterojunction diode fabricated by MBE. Near-band edge and deep-level emissions were observed from the LED devices at both low temperatures and room temperature, which is due to band-to-band and band-to-deep level radiative recombination in ZnO, respectively.In recent years, u et al.  reportedet al. [0.9Mg0.1O/ZnO/Zn0.9Mg0.1O/ZnO:Ga p-i-n heterostructures, which showed EL coming from deep level emission at low forward bias and near band edge ultraviolet emission at high bias. Ryu et al. [n-type and p-type ZnO and Be0.3Zn0.7O layers. Arsenic and gallium were used as dopants for p-type and n-type layers. The EL emission was attributed to the active layer. This kind of device structure was also used for laser diode [et al. [p-type ZnO/Ga-doped n-type ZnO with an MgZnO/ZnO/MgZnO quantum well embedded in the junction was grown on Si by molecular beam epitaxy. The diodes emit lasing at room temperature with a very low threshold injection current density of 10 A/cm2. The lasing mechanism is exciton-related recombination. Different devices of complex structure have been reported. Kim et al.  fabricatu et al.  fabricater diode . The las [et al.  also repc-axis oriented ZnO-based MOS (Au/SiOx(x < 2) /ZnO) device [In addition, ZnO based LEDs and laser diodes of MIS or MOS structure were also demonstrated, for example, a MIS structure LED , and the) device . et al. [p-type ZnO was formed by As+ ion implantation into the as-grown ZnO nanorods. Jeong et al. [n-ZnO/p-GaN heterojunction light-emitting diodes, showing electroluminescence emission of the wavelength of 386 nm come out from ZnO nanowire. Bao et al. [p-n junction diode has been produced using ZnO nanorods as the n-type material [et al. [p-type silicon substrate /i-ZnO-SiO2 nanocomposite/n-ZnO:Al heterojunction (p-i-n) laser diodes. The intrinsic layer, inserted between the n- and p-injection layers, consisted of ZnO powder embedded in a SiO2 matrix, prepared by the sol–gel technique. The lasing was due to the ZnO clusters.The random lasing action was also demonstrated by Leong and Yu  in p-SiCn-ZnO based hybrid LEDs: the p-GaN/n-ZnO:In (p-n) and p-GaN/i-ZnO/n-ZnO:In (p-i-n) heterojunction light-emitting diodes [It is noted from the above discussion that defects in ZnO, including undoped, manifest itself frequently in the luminescence. In order to fabricate ZnO based UV LED, ZnO of very few deep level defects has to be used for device fabrication. We have discussed above that the vapor cooling condensation method can deposit ZnO layer with much less oxygen vacancies. Using the method to deposit both doped and undoped ZnO layers, we have developed two kinds of g diodes . The strp-GaN/n-ZnO:In heterojunction LED consists of a broad emission band at 432 nm, which is attributed to the transition from the conduction band to the acceptor level in the Mg doped p-GaN when electrons are injected from the n-ZnO:In into the Mg-doped p-GaN. It is interesting that the EL spectrum of p-i-n device showed a pure UV band at 385 nm, which is consistent with the PL spectrum of the ZnO layer deposited with the vapor cooling condensation method. It indicates that the EL emission of the p-i-n LEDs comes from ZnO layer of the heterostructure, the undoped i-ZnO layer inserted between n-ZnO:In and p-GaN. It is due to the fact that the holes from p-GaN and electrons from n-ZnO:In are injected into the i-ZnO layer and radiatively recombined there. It is clear that the UV EL of thus developed p-i-n heterostructure is closely related to the fact that the inserted i-ZnO layer, deposited by the vapor cooling condensation method, has much less luminescent defects. It is also worth while to point out that the developed p-i-n LED is of potential applications in UV LEDs for high-temperature operation. [The room temperature current-voltage characteristics clearly demonstrated a rectifying diodelike behavior for both devices. The EL spectra of the fabricated p-n and p-i-n devices under forward bias are shown in eration. p-i-n UV LEDs [p-ZnO:AlN/i-ZnO/n-ZnO:In LED. The AlN codoped p-ZnO layer was deposited on sapphire substrates by using the radio frequency (RF) magnetron co-sputtering system with a dual RF power supply. The i-ZnO and n-type ZnO:In films were fabricated by the vapor cooling condensation method. The resultant LEDs showed a UV EL peaked at ~3.2 eV, operated under different injection currents at room temperature as shown in i-ZnO layer where the deep-level defects were very few as demonstrated previously. In a recent investigation, we succeeded in fabricating purely ZnO based  UV LEDs . Figure p-GaN layer/i-ZnO nanorod array/n-ZnO:In nanorod array heterostructured LEDs. Both i-ZnO and n-ZnO:In nanorod arrays in the structure were grown on a p-GaN layer through an anodic alumina membrane (AAM) template using the vapor cooling condensation method. We also succeeded in fabricating ZnO nanorod-based heterostructured ultraviolet LEDs . Figure p-i-n heterostructured LEDs operated at 15 μA and 35 μA. In particular, the p-i-n devices exhibit a UV EL emission centering at 386 nm, which becomes dominant emission at higher injection current of 35 μA. This UV band is attributed to the near-band-edge emission of the i-ZnO nanorod array of the p-i-n structure.Nanostructure can also been used to improve the light extraction efficiency. In a recent work of our laboratory , Al-dopeThe fabricated LEDs with AZO film prepared at a higher DC Al co-sputtering power (10 W) show an increase of 20% in the light output power compared to the conventional LEDs, which is 10% more than that of LEDs with AZO prepared at Al co-sputtering power of 7 W. The extra increase in the light output power was attributed to the formation of Al nanoclusters in AZO under higher Al sputtering power, which scattered the emitted light outward and hence increased the light extraction . ZnO related materials have been attracting intensive investigation due to inherent advantages and potential applications. In this paper, the recent progress on some aspects of the active research field, related to applications for the light-emitting diodes, has been reviewed in combination with the works carried out in our lab. One can feel that great progress has continuously been achieved, but there is still a long way to go for realizing extensive practical applications. Further efforts should be continued in various aspects of investigation for reaching the final goal.


N-isopropylacrylamide-co-itaconamic acid) p(NIPAM-co-IAM), which is formed by copolymerization of N-isopropylacrylamide (NIPAM) and itaconamic acid , was introduced into a solution of NIPAM to form a series of pH and thermo dual-responsive p(NIPAM-co-IAM)/pNIPAM semi-IPN hydrogels by free radical polymerization. The structural, morphological, chemical, and physical properties of the linear copolymer and semi-IPN hydrogels were investigated. The semi-IPN hydrogel showed high thermal stability according to thermal gravimetric analyzer (TGA). Scanning electronic microscopy (SEM) images showed that the pore size was in the range of 119~297 µm and could be controlled by the addition ratio of the linear copolymer in the semi-IPN structure. The addition of linear copolymer increased the fracture strain from 57.5 ± 2.9% to 91.1 ± 4.9% depending on the added amount, while the compressive modulus decreased as the addition increased. Moreover, the pH and thermo dual-responsive properties were investigated using differential scanning calorimetry (DSC) and monitoring the swelling behavior of the hydrogels. In deionized (DI) water, the equilibrium swelling ratio of the hydrogels decreased as the temperature increased from 20 °C to 50 °C, while it varied in various pH buffer solutions. In addition, the swelling and deswelling rates of the hydrogels also significantly increased. The results indicate that the novel pH-thermo dual-responsive semi-IPN hydrogels were synthesized successfully and may be a potential material for biomedical, drug delivery, or absorption application.A series of semi-interpenetrating polymer network (semi-IPN) hydrogels were synthesized and investigated in this study. Linear copolymer poly( N-isopropylacrylamide) (pNIPAM) is the most widely investigated.Hydrogel is a type of polymer material with a three-dimensional network capable of absorbing water without dissolving. It can be molded into any form, shape, or size for many potential applications in many fields. In recent times, smart hydrogel has been studied extensively due to its phase transitions in response to external stimulus, including temperature, pH, ionic strength, light, pressure, redox, and electric field ,7,8,9,10pNIPAM exhibits a lower critical solution temperature (LCST), about 32 °C in pure water, which is close to human body temperature ,12,13. AFor several potential applications, such as drug delivery and absorbing systems, a pH and thermo dual-responsive hydrogel with a high swelling ratio, a fast response rate, and good mechanical properties is needed, because temperature and pH are important environmental factors in these systems. One of the most useful methods to meet these requirements is to prepare semi-interpenetrating (semi-IPN) hydrogels by introducing a high hydrophilic linear polymer into the pNIPAM network. Semi-IPN hydrogels are usually synthesized by simultaneous polymerization of a monomer system with a cross-linking agent in the presence of natural polymer ,25,26,272 and –COOH)  was 10% /poly(N-isopropylacrylamide) system [N-isopropylacrylmaide) system [N,N-dimethylacrylamide-co-2-hydroxyethyl methacrylate) system [Although it had the highest values of compressive modulus and fracture stress, the conventional pNIPAM hydrogel showed the lowest fracture strain, 57.5 ± 2.9%. Meanwhile, the semi-IPN hydrogels had a fracture strain of 71.2 ± 3.5 and 91.1 ± 4.9% for PNA1 and PNA4 samples, respectively. The results demonstrate that linear copolymer p(NIPAM-co-IAM) increases the fracture strain of semi-IPN hydrogels. The effect of the linear copolymer on fracture strain can be explained by its high viscosity, which helps energy dissipation take place rapidly during deformation. In addition, the semi-IPN hydrogels were highly hydrophilic, which helped the water to be more stable in the network, leading to significantly increased fracture strain, because water acted as a plasticizer . Similar) system , salecan) system , and sal) system . The aboThe thermo-sensitive property of the semi-IPN hydrogels is demonstrated in Besides being thermo-responsive, the semi-IPN hydrogels also showed pH-responsive properties. 2 are readily ionized to –COO− and NH3+. In the pH 7 buffer solution, the negatively and positively charged moieties were probably balanced and the hydrogel system reached the isoelectric point. At this point, the repulsion of moieties with the same type of charge almost disappeared, while the attraction of –COO– and NH3+ became strong and shrinkage took place in the hydrogel network, leading to the lowest swelling ratio. In the acidic buffer solutions , the functional groups –NH2 were ionized into –NH3+, leading to a greater quantity of positive charges than negative charges. On the contrary, in the pH 8 buffer solution, the –COOH groups were ionized into –COO–, resulting in a greater quantity of negative charges than positive charges. In both cases, the hydrogel networks expanded rapidly by electrostatic repulsion, leading to increased swelling ratios. However, as the pH value went up from 8 to 10, the swelling ratios reduced slightly. This may be because the existence of alkaline ions in the swelling environment with high concentration caused a “charge screening effect” [– groups. This caused the polymer structure to come closer and reduced expansion, leading to decreased swelling ratios. The mechanism for the influence of pH values on the swelling ratios of the hydrogels is illustrated in It was reported that the swelling ratios of hydrogel are influenced by the isoelectric point and the repulsion of moieties with the same type of charge in the material system. First of all, it is necessary to assume that in the aqueous solution, the functional groups –COOH and –NH effect” . These p1H NMR, suggests that linear polymer chains were entangled in pNIPAM networks. In addition, TGA, rheological, and mechanical tests were measured, indicating thermal stability, viscoelastic behavior, and good mechanical properties of the semi-IPN hydrogel. SEM images showed a uniform porous structure of the hydrogels with pore size ranging from 119 ± 16 to 297 ± 61 µm. Moreover, the LCST and swelling properties of the hydrogels in DI water and buffer solution with pH ranging from 4 to 10 at different temperatures were measured. The LCST of the semi-IPN hydrogels in DI water increased slightly from 31.6 to 32.7 °C with increased linear copolymer content, while in pH buffer solution it was in the range of 25.2 to 34.7. The swelling ratios were influenced by linear copolymer content and pH environment: increased linear copolymer content or acid/base solution increased the swelling ratio. The swelling and deswelling rates of the semi-IPN hydrogels were also significantly accelerated with increased linear copolymer content. The phase transition and swelling behavior of the semi-IPN hydrogel in this study can be significantly changed by a change of temperature and/or pH environment, indicating that it can potentially be used for a drug delivery system or as an absorbing material.In this work, a novel p(NIPAM-co-IAM)/pNIPAM semi-IPN hydrogel based on NIPAM and IAM monomers was prepared by free radical polymerization. By introducing the linear copolymer p(NIPAM-co-IAM) into a solution of NIPAM monomer and MBA cross-linker, pH and thermo dual-responsive semi-IPN hydrogels were obtained. First, GPC and DLS tests showed that the IAM co-monomer reduced the molecular weight and increased the hydrodynamic diameter of the linear copolymer. The chemical structure, confirmed by FTIR and